DESCRIPTION (based on applicant's abstract): Drug-resistant strains of Plasmodium falciparum are relentlessly emerging throughout the world; they have emerged especially rapidly in Southeast Asia. As a result, the artemisinin derivatives are now used widely in Thailand and neighboring countries. We will test the following three hypotheses: (1) resistance to artemisinin compounds will emerge in Indochina; a sentinel surveillance system established in this area will allow its early detection and control; (2) low- levels of artemisinin resistance are already present in some areas of Thailand. Recrudescence after artesunate-mefloquine treatment is associated with in vitro artemisinin insensitivity; and (3) in vitro chloroquine, mefloquine, quinine, and antifolate resistance and artemisinin insensitivity are associated with specific genetic polymorphisms. We will expand upon an already established sentinel surveillance system for drug-resistant malaria in Thailand and bordering countries. AFRIMS personnel will regularly visit malaria clinics in endemic areas as well as conduct malaria surveys. Blood will be drawn from patients (upon initial visits, and, in some cases, on recrudescence) and used to establish in vitro cultures. These isolates (as well as clones made from these isolates) will then be tested for sensitivity to chloroquine, quinine, mefloquine, antifolates and artemisinin derivatives. DNA from these isolates will be sent to Toronto, where they will be assessed for DNA sequence polymorphisms in the dihydrofolate reductase (DHFR), dihydropteroate synthase (DHPS), cg2, and pfmdr1 genes. Isolates and clones will be sent to Michigan where they will be evaluated for their abilities to take up and metabolize drug and analyzed for polymorphisms in artemisinin target proteins, such as the Transitionally Controlled Tumor Protein (TCTP). Attempts will then be made to correlate these biomarkers with in vitro and in vivo drug sensitivities. The proposed study will be one of the first large- scale molecular epidemiological investigations of drug resistance in malaria and may greatly add to our ability to identify and control drug-resistant malaria.